CN115894661B - Antibacterial peptide and application thereof - Google Patents
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- CN115894661B CN115894661B CN202210959941.2A CN202210959941A CN115894661B CN 115894661 B CN115894661 B CN 115894661B CN 202210959941 A CN202210959941 A CN 202210959941A CN 115894661 B CN115894661 B CN 115894661B
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- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 19
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 12
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 12
- 241000251468 Actinopterygii Species 0.000 claims description 9
- 239000003674 animal food additive Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 208000035143 Bacterial infection Diseases 0.000 claims description 4
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 25
- 241000252230 Ctenopharyngodon idella Species 0.000 abstract description 21
- 108010074108 interleukin-21 Proteins 0.000 abstract description 18
- 102100030704 Interleukin-21 Human genes 0.000 abstract description 14
- 230000004071 biological effect Effects 0.000 abstract description 6
- 229940088710 antibiotic agent Drugs 0.000 abstract description 5
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- 102000015696 Interleukins Human genes 0.000 abstract 1
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- 238000011481 absorbance measurement Methods 0.000 description 15
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- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 229920001184 polypeptide Polymers 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
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- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 4
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
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- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Peptides Or Proteins (AREA)
Abstract
The application provides an antibacterial peptide and application thereof, and relates to the technical field of biological antibacterial. According to the application, the grass carp interleukin 21 sequence is modified based on the grass carp interleukin 21 sequence directly extracted from animal and plant individuals, so that the antibacterial peptide is obtained, the raw material sources are more scientific and wide, the manufacturing period is shortened, the manufacturing cost is reduced, the yield is improved, the grass carp interleukin 21 sequence is modified aiming at the defects of lower biological activity and poor stability of the natural antibacterial peptide, and the structure is optimized to increase the biological activity and the stability of the grass carp interleukin 21 sequence, so that the antibacterial capability and the application value of the grass carp interleukin are improved, and the antibacterial peptide gradually replaces antibiotics for treating fish diseases, thereby conforming to the trend of environment-friendly and pollution-free cultivation, and having important practical and strategic significance.
Description
Technical Field
The invention relates to the technical field of biological antibiosis, in particular to an antibacterial peptide and application thereof.
Background
Grass carp is one of four large fishes cultivated in Chinese fresh water, and has important economic value. However, grass carp has low disease resistance and survival rate, is easy to suffer from hemorrhagic disease, gill rot, red skin disease, enteritis and the like, and increases the probability of pathogen cross infection among aquatic animals by high-density cultivation, so that the diseases are increasingly aggravated. However, the conventional method for preventing and treating fish diseases, which is a large amount of antibiotics, has adverse effects on abuse, such as reduced quality of aquatic products, increased drug residues in the environment and increased bacterial resistance, so that it is highly demanded to find a drug capable of replacing antibiotics.
As a novel antibacterial drug, the Antibacterial Peptide (AP) has the characteristics of small molecular weight, good water solubility, wide antibacterial spectrum, special action mechanism, difficulty in generating drug resistance and the like, can inhibit viruses, fungi and parasites to a certain extent, and becomes a 'tie-up' research hotspot. Because of the large number of microorganisms in the aquatic environment in which fish live, antimicrobial peptides are an important component of fish innate immunity. However, the natural antibacterial peptide has the defects of low biological activity and poor stability, so the natural antibacterial peptide needs to be modified, and the biological activity and the stability of the natural antibacterial peptide are improved by optimizing the structure of the natural antibacterial peptide, so that the antibacterial capability and the application value of the natural antibacterial peptide are improved, and the antibacterial peptide gradually replaces antibiotics for treating fish diseases, thereby being in line with the trend of environment-friendly and pollution-free cultivation and having important practical and strategic significance.
Disclosure of Invention
The present invention aims to provide an antibacterial peptide and application thereof, so as to improve the problems. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present application provides an antimicrobial peptide comprising:
The antibacterial peptide is obtained based on modification of the amino acid sequence of a grass carp interleukin 21 gene, wherein the amino acid sequence of the grass carp interleukin 21 gene is SEQ ID No.1, and the amino acid sequence of the antibacterial peptide is SEQ ID No.2.
In a second aspect, the application also provides a medicament for treating bacterial diseases of fish, which comprises the antibacterial peptide.
In a third aspect, the present application also provides an antibacterial agent comprising the above antibacterial peptide.
In a fourth aspect, the application also provides a feed additive, which is characterized in that the feed additive comprises the antibacterial peptide.
The beneficial effects of the invention are as follows:
On the one hand, the application modifies the grass carp interleukin 21 sequence based on the grass carp interleukin 21 sequence directly extracted from animals and plants, thereby obtaining the antibacterial peptide, not only having more scientific and wide raw material sources, shortening the manufacturing period, reducing the manufacturing cost and improving the yield, but also modifying the grass carp interleukin 21 sequence aiming at the defects of lower biological activity and poor stability of the natural antibacterial peptide, and improving the biological activity and the stability of the grass carp interleukin 21 sequence by optimizing the structure, so as to improve the antibacterial capability and the application value of the grass carp interleukin 21 sequence, so that the antibacterial peptide gradually replaces antibiotics for treating fish diseases, thereby conforming to the environment-friendly and pollution-free cultivation trend and having important practical and strategic significance.
On the other hand, the antibacterial peptide obtained in the application can be applied to medicines for treating bacterial diseases of fishes, and can be used for treating diseases such as bacterial enteritis of grass carp and the like; can also be used as a feed additive, thereby improving the survival rate of fish; can also be used as antibacterial agent, has wide antibacterial spectrum, is not easy to generate drug resistance, and can also resist fungi and viruses.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the embodiments of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims thereof as well as the appended drawings.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and should not be considered as limiting the scope, and that other related drawings can be obtained according to these drawings without inventive effort for a person having ordinary skill in the art.
FIG. 1 is antibacterial peptide information for positive control;
FIG. 2 is a data analysis chart of the amino acid sequence M4 of the antimicrobial peptide analyzed by CAMP R3;
FIG. 3 is a data analysis of the amino acid sequence M4 of the antimicrobial peptide by bioinformatics tools;
FIG. 4 is a statistical chart of the results of the first absorbance measurement experiment;
FIG. 5 is a statistical chart of the results of the second absorbance measurement experiment.
FIG. 6 is a graph of colony growth in a plate assay;
FIG. 7 is a statistical plot of colony counts for the plate experiments.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by one of ordinary skill in the art without inventive effort, are within the scope of the present invention based on the embodiments of the present invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures. Meanwhile, in the description of the present invention, the terms "first", "second", and the like are used only to distinguish the description, and are not to be construed as indicating or implying relative importance.
The invention provides an antibacterial peptide which is obtained by modifying the amino acid sequence of a grass carp interleukin 21 gene, wherein the amino acid sequence of the grass carp interleukin 21 gene is SEQ ID No.1, and the amino acid sequence of the antibacterial peptide is SEQ ID No.2.
The method for modifying the antibacterial peptide comprises the following steps:
and S1, antibacterial area sequence analysis. The sequence of grass carp interleukin 21 is analyzed by using a bioinformatics tool CAMP R3, the antibacterial capability of the sequence is firstly predicted, and then a sequence fragment with antibacterial property is predicted and obtained.
And S11, analyzing the antibacterial capacity of the grass carp IL-21 amino acid sequence by using CAMP R3. The known grass carp IL-21 amino acid sequence is input into CAMP R3 software, and the Predict Antimicrobial Peptides option of the AMP Prediction tool in the page is selected to predict the antibacterial capability of the sequence. To more accurately and efficiently identify the fragment having antibacterial peptide properties in the IL-21 amino acid sequence, the sequence fragment IIKSLKSISNESRKKV having antibacterial properties in the IL-21 amino acid sequence was then predicted using Predict Antimicrobial region WITHIN PEPTIDES in the AMP Prediction tool.
Step S12, carrying out physicochemical property analysis on the sequence fragment IIKSLKSISNESRKKV obtained by the analysis in step S1 by using HeliQuest and ProtParam. The main function of HeliQuest is to analyze and screen alpha-helical peptide, and the main function of ProtParam is to analyze physicochemical properties of peptide sequence. The method comprises the steps of attempting to carry out point mutation (mutation for enhancing hydrophobicity: tryptophan (W) and mutation for enhancing electronegativity: lysine (K)) on an amino acid sequence, and synthesizing data of a series of mutant antibacterial peptides to obtain the antibacterial peptide with optimal data, wherein the method is specifically characterized in that the 9 th amino acid S and the 11 th amino acid S of a original sequence are mutated into W, and the 12 th amino acid R is mutated into K, namely IIKSLKSIWNEWKKKV. And combining the sequence after point mutation with positive control polypeptide and deleting part of amino acid sequence according to the three-dimensional structure obtained by PEP-FOLD3 analysis, wherein the specific expression is that the positive control polypeptide sequence, namely IIKSLKSIWNEWKKKVIGGIISFFKRLF, is added at the C end of the sequence after point mutation, and the 15 th to 19 th amino acid and the 26 th to 28 th amino acid are deleted to obtain the final amino acid sequence IIKSLKSIWNKWKKIISFFK. Finally, the Antimicrobial ability of amino acid sequence IIKSLKSIWNKWKKIISFFK is analyzed by using bioinformatics tools such as CAMP R3, heliQuest, AMP Prediction, antimibeal PEPTIDE SCANNER vr.2, axPEP, PEP-FOLD3 and the like, the closer the score data is to 1, the stronger the potential Antimicrobial ability is indicated by the score data, the more specific results of the analysis are shown in FIG. 2 and FIG. 3, FIG. 2 is a data analysis chart of the Antimicrobial peptide amino acid sequence M4 analyzed by CAMP R3, and FIG. 3 is a data analysis chart of the Antimicrobial peptide amino acid sequence M4 analyzed by bioinformatics tools. After the step is successfully carried out, the antibacterial peptide with potential antibacterial capability is obtained, and the sequence is SEQ ID No.2 in a sequence table and is named as M4.
Step S2, verifying the function of M4 by an antibacterial experiment: the antibacterial ability of the antibacterial peptide obtained in step S12 was verified by absorbance measurement experiment and plate experiment, respectively, and compared and analyzed with the antibacterial ability of the positive control antibacterial peptide, the detailed information of the positive control antibacterial peptide used in this example is shown in fig. 1, and fig. 1 is the antibacterial peptide information of the positive control.
Step S21, absorbance measurement experiment.
Step S211, absorbance measurement experiment principle.
Since the absorbance, that is, the OD value, represents the optical density absorbed by the object to be detected, the concentration of the cells in the bacterial liquid can be estimated by detecting the absorbance of the bacterial liquid, thereby reflecting the growth state. If antibacterial peptide is adopted to treat bacteria in the process, if the antibacterial peptide is effective, the OD value of the bacterial liquid is reduced, and if the antibacterial peptide is ineffective, the OD value of the bacterial liquid is not obviously different from the OD value of the control bacterial liquid, so that the antibacterial capability of the antibacterial peptide can be evaluated through the determination of the absorbance value.
Step S212, absorbance measurement experiment design.
Step S2121, the antibacterial peptides used in the experiment are all synthesized by Jier biosystems and stored in a refrigerator at-80 ℃. In application, M4 and positive antibacterial peptide PC are taken out from-80deg.C refrigerator and placed on ice box temporarily. The antimicrobial peptides were centrifuged in a bench top high-speed refrigerated centrifuge at 4℃and 12000rpm for 10min. Then, 1mL of distilled water (1 mg/mL) was added to each of the antimicrobial peptides, and vortexed and shaken for 5s to allow the antimicrobial peptides to be sufficiently dissolved.
Step S2122, the experimental fungus is aeromonas hydrophila isolated, purified and stored in a comparative immunology laboratory of university of electronic technology. And taking out the test tube containing the aeromonas hydrophila bacterial liquid from the ultralow temperature refrigerator and placing the test tube on an ice box. 5. Mu.L of the bacterial liquid was extracted into a clean BD tube under sterile conditions, and 5mL of TSB medium solution was added. After thoroughly mixing, BD tube was placed in a thermostatic incubator shaker at 30℃and 180rpm for overnight incubation.
Step S2123, taking 600 mu L of TSB culture medium solution to two 1.5mL EP tubes under aseptic conditions, adding the culture medium solution in the EP tubes into a cuvette, placing the cuvette in a double-beam ultraviolet-visible spectrophotometer, and calibrating the instrument. Taking out overnight culture Aeromonas hydrophila liquid, taking 600 mu L of Aeromonas hydrophila liquid under aseptic condition to measure OD value of the liquid, and diluting the liquid to about 1OD (bacterial concentration is about 109 CFU/mL) with culture medium solution according to the measured value.
Step S2124, first absorbance measurement experiment.
The bacteria and antimicrobial peptides were mixed uniformly in a sterile environment in the following groups:
The negative control group system is aeromonas hydrophila bacterial liquid and distilled water; the positive control group is Aeromonas hydrophila bacterial liquid+5mg/mL positive antibacterial peptide solution; the antibiotic group is aeromonas hydrophila bacterial liquid and gentamicin solution; the incubation system is 50 mu L of aeromonas hydrophila bacteria liquid/PBS solution+50 mu L of gentamicin/antibacterial peptide/distilled water; the experimental group was Aeromonas hydrophila broth+three concentration gradients (1 mg/mL, 0.1mg/mL, 0.01 mg/mL) of M4, each set with 3 replicates.
After the OD value of 0h is measured at 600nm by a full-wavelength enzyme-labeled instrument, the 96-well plate is put into a constant-temperature oscillating table, incubated at 30 ℃, OD value measurement is carried out at intervals, and the antibacterial function of the antibacterial peptide is judged according to the OD value.
Step S2125, second absorbance measurement experiment.
To determine the Minimum Inhibitory Concentration (MIC) of M4 and to better compare the antimicrobial effect of M4 and PC at the same mass concentration, the following modifications were made to the experimental group:
The positive control group is Aeromonas hydrophila bacterial liquid+1mg/mL positive antibacterial peptide solution; the experimental group is Aeromonas hydrophila bacterial liquid plus three concentration gradients (1 mg/mL, 0.3mg/mL, 0.1 mg/mL) of M4; the remaining conditions were the same as in the first absorbance measurement experiment.
Step S2126, analyzing the absorbance measurement experiment result.
The results of the two absorbance measurement experiments are shown in fig. 4 and 5, wherein fig. 4 is a statistical chart of the results of the first absorbance measurement experiment, and fig. 5 is a statistical chart of the results of the second absorbance measurement experiment. In the first absorbance measurement experiment, the OD value of the antibacterial peptide group is continuously increased from 0 hour, and the OD value is slowly increased after 16 hours and reaches the plateau phase. The OD value of the M4 peptide at the concentration of 0.01mg/mL and 0.1mg/mL is not greatly different from that of the negative control group, the initial absorbance is larger than that of the negative control group at the concentration of 1mg/mL, the final concentration is smaller than that of the negative control group, and the positive control group at the larger concentration (5 mg/mL) is also similar to that of the positive control group. The OD values of the antibiotic group were essentially unchanged. In the second absorbance measurement experiment, the M4 peptide showed a decrease in OD value within 4 hours at concentrations of 1mg/mL and 0.3mg/mL, followed by an increase, reached a plateau in OD value increase after 21 hours, and the initial OD value was higher than that of the negative control group, and the final OD value was lower than that of the negative control group. The change trend of OD value of the M4 peptide at 0.1mg/mL is not greatly different from that of the negative control group, and the same is true of the positive polypeptide at 1 mg/mL. Although there is no trend of decreasing the OD value, the positive control group of 1mg/mL M4 and 5mg/mL is smaller than the negative control group in comparison with the increasing value of the OD value, and the effect of inhibiting the bacterial growth of the M4 and the positive control polypeptide can be demonstrated. In the third measurement, by comparing the increase value of OD, the increase value of M4 is smaller than that of the negative control group at the concentrations of 1mg/mL and 0.3mg/mL, and the difference between the positive polypeptide and the negative control group is not large at the concentration of 0.1mg/mL, and the positive polypeptide with the concentration of 1mg/mL is also the same, so that the minimum antibacterial concentration is 0.3mg/mL, and meanwhile, compared with the positive control polypeptide and the M4, the antibacterial effect of the obvious M4 at the same concentration is better.
Step S22, plate experiments.
Step S221, a plate experiment principle.
In the plate experiment, aeromonas hydrophila and the antibacterial peptide can be adopted to incubate together and then coated on a solid plate, and the colony number is compared after the culture at a proper temperature, so that the antibacterial capability of the antibacterial peptide is compared.
Step S2211, design of a flat experiment.
Step S2212, wherein the experimental group is M4 with the concentration of 1mg/mL, the negative control group is Aeromonas hydrophila liquid+ddH 2 O, the positive control group is Aeromonas hydrophila liquid+5 mg/mL positive antibacterial peptide solution, and each group is repeated 4 times. The antibiotic group is aeromonas hydrophila bacterial solution and gentamicin solution. The incubation system was 50. Mu.L of Aeromonas hydrophila/PBS solution+50. Mu.L of gentamicin/antibacterial peptide/distilled water, and mixed well.
And (3) placing the incubation system into a constant-temperature shaking table, and incubating at 30 ℃ for 30min to enable the aeromonas hydrophila bacterial solution and the antibacterial peptide solution to be fully contacted. Igniting the alcohol lamp, cleaning the inoculating loop by alcohol solution beside the alcohol lamp, burning on the flame outer flame of the alcohol lamp, and then cooling aside. 100. Mu.L of the mixed bacterial liquid was taken out of each incubation system by a pipette and spread on a TSA solid medium, and incubated in a 30℃incubator for 28 hours, and colony counting was performed.
Step S2213, analyzing the plate experiment result.
Typical colony growth patterns and colony count patterns are shown in fig. 6 and 7, fig. 6 is a colony growth pattern of a plate test, fig. 7 is a colony count pattern of a plate test, and the colony count M4 of each group is obviously smaller than that of a negative control group, so that it can be shown that M4 has an antibacterial effect.
On the other hand, the antibacterial peptide obtained in the application can be applied to medicines for treating bacterial diseases of fishes, and can be used for treating diseases such as bacterial enteritis of grass carp and the like; can also be used as a feed additive, thereby improving the survival rate of fish; can also be used as antibacterial agent, has wide antibacterial spectrum, is not easy to generate drug resistance, and can also resist fungi and viruses.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations of the present invention will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art can easily think about variations or substitutions within the technical scope of the present invention, and the scope of the present invention is intended to be covered by the present invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.
Claims (4)
1. An antimicrobial peptide, characterized in that: the amino acid sequence of the antibacterial peptide is SEQ ID No.2.
2. A medicament for the treatment of bacterial diseases in fish, comprising the antibacterial peptide according to claim 1.
3. An antibacterial agent comprising the antibacterial peptide according to claim 1.
4. A feed additive comprising the antibacterial peptide according to claim 1.
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| CN108752457A (en) * | 2018-05-23 | 2018-11-06 | 华中农业大学 | A kind of derived peptides and its application from grass carp interferon |
| CN108949598A (en) * | 2018-09-13 | 2018-12-07 | 浙江皇冠科技有限公司 | Recombinate the engineering bacteria of Fish leukocyte interleukin -2 and its preparation method of product |
| CN113754750A (en) * | 2021-09-30 | 2021-12-07 | 华中农业大学 | Antibacterial peptide and application thereof in aquaculture |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20010023070A1 (en) * | 1998-05-29 | 2001-09-20 | Reinhard Ebner | Interleukins-21 and 22 |
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